P1380: THE COMPOSITION OF INFUSED CD19-CAR-T PRODUCT IMPACTS ON EFFICACY
Authors: Silvia Bellesi, Eugenio Galli, Giacomo DI Cesare, Camilla Iacovelli, Ilaria Pansini, Elena Maiolo, Eleonora Alma, Rosalia Malafronte, Flaminia Bellisario, Francesco D’ Alò, Maria Bianchi, Nicola Piccirillo, Patrizia Chiusolo, Simona Sica, Federica Sorà, Stefan Hohaus
Journal: HemaSphere (2023)

Topic: 24. Gene therapy, cellular immunotherapy and vaccination - Biology & Translational Research Background: The use of CD19-CAR-T has become the standard therapy for several R/R B-cell malignancies. Definition of outcome predictors, such as pts and disease-related risk factors (e.g. lymphocyte phenotype at apheresis, lymphodepletion, tumor burden) are still incomplete. Recently, CD8+CAR+ with a central memory (CM) phenotype in the product have been associated to higher peak of CAR-T at day 10, which was predictive for better outcome (Mofrini et al, 2022). Aims: We investigated the CAR-T product in terms of CD4/CD8 CAR+ ratio, CAR-T-cell subset, cellular activated phenotype. Methods: We analyzed leftovers from CAR-T bags. CAR-T cells were detected with a human CD19 His Tag protein and an anti-His APC-labelled secondary antibody. CD45RA and CD62L were used to define CAR-T subset as: naïve (N), CM, effector memory (EM), and terminal effector memory (TEMRA). The co-expression of CD38 and HLA-DR defines activated cells. Data were acquired on BDFACS Canto II Cytometer and analysed with Diva Software. Results: We included 31 pts with aggressive B-lymphoma, treated with tisa-cel (13), axi-cel (13) or brexu-cel (5). Median age, previous lines of therapy, disease status at infusion and LDH levels were similar among groups. The percentage of CAR+CD3+ was significantly lower in the tisa-cel product when compared to axi-cel and brexu-cel (45% vs 71% vs 79%, p=0.022). The percentage of CAR+CD8+ was higher in Brexu-cel than others (60.5% vs 26-38%, p 0.03). In all products the most representative T subset was EM both for CAR+CD4+ and CAR+CD8+ (around 87%). The vast majority of CAR+CD8+ displayed an activated phenotype with a slightly higher proportion in tisa-cel (80.3%) in comparison to axicel (70.9%) and brexu-cel (75.9%) bags [p=0.052]. Activated CAR+CD8+ were lower in pts with more than 2 previous lines (p=0.025). We next analyzed the impact of CAR-T phenotypes on outcome. We considered as responders pts obtaining CR/PR at one, three and six months (M1 M3 and M6) after CAR-T infusion, and compared them with pts failing to achieve any response at the same time points (non-responders). Pts with early failure at M1 showed extremely low proportion of CAR+CD8+EM cells in infused bags compared to responders (5.1% vs 91.8%, p=0.057). The probability of survival of these pts at M3 was low. Infused bags from responders at M3 and M6 showed a higher percentage of CAR+CD8+ cells than non-responders (56% vs 27% p=0.02 and 55.4% vs 27.3% p 0.034 respectively). As a consequence, we observed significant difference in the ratio CD4+/CD8+ CAR+ in infused bags between responders at M3 vs non-responders (ratio 0.74 vs 2.47, p=0.011) (Figure 1A) We used a ROC analysis to identify an ideal ratio CD4+/CD8+ CAR+ cut-off among infused CAR-T to discriminate responders at M3: we found that a ratio of 1.12 had 100% sensitivity and 70 % specificity in identifying responses at M3 (AUC 82%, p=0.001). (Figure 1B) In a logistic regression analysis, the CD4+/CD8+ CAR+ ratio was confirmed as a variable to predict response at M3 (Odds Ratio 23.3 p=0.012) and M6 (Odds Ratio 10 p=0.028). Summary/Conclusions We found that a higher percentage of CAR+CD8+ T cells and a lower ratio CD4+/CD8+ CAR+ in the infusion bags are significant predictors for response at 3 and 6 months in pts treated with CD19-CAR-T. We observed that a very low percentage of CAR+CD8+EM T cells may be associated with early treatment failure at 1 month. These data support the role of CD8-mediated cytotoxic mechanism of action by CAR-T cells, and may be useful for strategies of potential empowerment of manufactured products in order to raise CAR-T efficacy.Keywords: Flow cytometry, Immunophenotype, CAR-T